Research in the Frederich Lab focuses on the chemistry and biology of natural products that modulate protein-protein interactions (PPIs). Our long-term objective is to understand and enhance the pharmacology of our targets using chemical synthesis. In this proposal, we investigate a family of complex diterpene glycosides with demonstrated efficacy for 14-3-3 PPIs in human cell culture. 14-3-3 proteins are a family of adapter molecules that regulate several hundred client proteins (CPs) by forming binary protein complexes. This expansive 14-3-3 interactome is integrated into the phosphorylation- depended signaling pathways that regulate cellular homeostasis. Dysregulation of 14-3-3/CP interactions has been implicated in the development of cancer and neurological disorders. Thus, small-molecules targeting 14- 3-3 functions harbor special potential as tools to interrogate 14-3-3/CP interactions in biochemical pathways. They also provide entry to lead structures for the development of new therapy modalities. The diterpene glycosides fusicoccin A (FC) and cotylenin A (CN) are archetypal fusicoccanes that stabilize 14-3-3 PPIs in vivo. FC engages a select set of 14-3-3/CP interactions and prolongs the lifetime of these PPIs by forming contacts with both proteins. This biology seeded the development of fusicoccin-THF, a semi- synthetic analog of FC with peripheral structural modifications that alter binding affinity and selectivity for 14-3- 3 PPIs. These observations suggest to us that the shared carbocyclic nucleus of FC and CN is a privileged motif for the design of selective 14-3-3 PPI stabilizers. However, entry to this substructure is hampered by its stereochemical complexity and the only existing means to access FC and CN is from producing fungi. We propose to use chemical synthesis as a tool to harness the potentially impactful pharmacology of these natural products. The objective of Aim 1 is to establish a synthetic blueprint for the rapid and modular assembly of FC and CN. In preliminary studies, we have developed photochemistry to flexibly prepare the carbocyclic core of our targets. In Aim 1, we will adapt and extend this chemistry to complete, for the first time, fully synthetic access to these diterpene glycosides. The objective of Aim 2 is to test whether the FC nucleus can scaffold 14-3-3 PPI stabilizers with superior selectivity profiles. In support of this work we have established biophysical tools to assay functional 14-3-3/CP interactions in the presence or absence of synthetic compounds. Guided by computational models, we propose custom syntheses of ligands targeting three regulatory 14-3-3 PPIs involved in cancer biology. The proposed research is significant to human disease because it will provide refined molecular tools to modulate 14-3-3 functions that modify disease pathways. This work is significant to fundamental chemical science because it will establish a versatile strategy to prepare fusicoccanes with diverse functional properties. This proposal is innovative because stabilizing protein complexes is an underexplored strategy in small-molecule PPI modulation.